Taylor J M, Tse T P
J Biol Chem. 1976 Dec 10;251(23):7461-7.
Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.
通过多核糖体免疫沉淀和聚（U）-琼脂糖亲和层析，已将大鼠肝脏白蛋白信使核糖核酸纯化至表观均一性。通过双抗体技术从总肝脏多核糖体中分离出合成白蛋白的特异性多核糖体，该技术可分离出特异性免疫沉淀物。将白蛋白-多核糖体免疫沉淀物溶解在去污剂中，并通过蔗糖梯度离心将多核糖体核糖核酸与蛋白质分离。然后通过聚（U）-琼脂糖与信使核糖核酸的聚腺苷酸3'末端结合，通过亲和层析从核糖体核糖核酸中分离出白蛋白信使核糖核酸。纯白蛋白信使核糖核酸在含85%甲酰胺的线性蔗糖梯度上以18S峰迁移，在十二烷基硫酸钠中的2.5%聚丙烯酰胺凝胶上以22S峰迁移。当将其添加到源自兔网织红细胞裂解物或小麦胚芽提取物的异源无细胞蛋白质合成系统中时，它编码真正的肝脏白蛋白的翻译。在网织红细胞裂解物中的翻译分析表明，白蛋白多核糖体从总肝脏多核糖体中纯化了约9倍，而白蛋白信使核糖核酸从白蛋白多核糖体核糖核酸中纯化了约74倍。通过凝胶电泳和胰蛋白酶肽图谱分析，在依赖信使核糖核酸的小麦胚芽系统中加入纯信使核糖核酸后，总翻译产物被鉴定为真正的白蛋白。
